The Myc family of transcription factors play key roles in cell proliferation, differentiation and cell death (apoptosis). However, deregulated expression of Myc family members, such as that which results from gene amplification or chromosomal translocation as observed in a variety of cancers, can have dire consequences. This is no more clearly illustrated than by the relationship between the amplification of the N- myc oncogene and patient outcome in childhood neuroblastoma. Neuroblastoma accounts for 8-10% of all childhood malignancies. In the context of Phase I SBIR funded research, we have screened a 34,000 small molecule library for inhibitors of N-Myc using a cell based readout system. We identified a series of hits representing distinct structural classes that demonstrated strong growth inhibitory effects against Myc- expressing cells compared to cells lacking any Myc family member. These hits are also reasonably specific at the transcriptional level since they were much more effective (3-6 fold) against N- and c-Myc mediated transactivation than against other E-box-targeted transcription factors. In addition, these hits showed strong activity against neuroblastoma cells but not normal cells (approximately 3-5 fold more sensitive). These results provide proof-of-principle that N-Myc inhibitors with desirable properties can be identified. The goal of this proposal is to perform structure-activity relationship (SAR) studies using focused libraries generated around the top 5 structurally distinct hits from the Phase I screening in order to identify more potent and specific advanced hits. Based on SAR analysis, the most potent and structurally "druggable" molecules from a minimum of 3 classes will be evaluated for their toxicity, pharmacokinetic properties and efficacy in vivo using a murine neuroblastoma model. Successful completion of this proposal will enable us to identify chemical structures that will serve as the basis for the hit-to-lead optimization phase of this project such that we can identify N- Myc inhibitors with the best biological activity and physiochemical properties that will have the greatest potential for successful use as targeted drugs for the treatment of childhood neuroblastoma. [unreadable] [unreadable] [unreadable]